The cAMP-dependent protein kinase holoenzyme (PKA) is composed of a two monomeric catalytic (C) subunits, which are released and activated when cAMP binds to the dimeric regulatory (R) subunit. The PKA C-subunits are prototypes for the kinase enzyme class, which covalently modify several types of protein substrates via a phosphorylation mechanism. One general type of phosphoprotein is rate-limiting enzymes of metabolism, which are modified by phosphorylation. Another general type of phosphoprotein substrate regulates more sustained effects through the regulation of gene transcription. These proteins are either induced by and/or phosphorylated by a PKC C-subunit mediated mechanism. At least three distinct C-subunit isozymes, designated C-alpha, C-beta, and C-gamma, carry out these cAMP- regulated responses. Based on sequence homology, C-gamma is clearly a PKA C-subunit. Recently, a rabbit anti-bovine heart C-subunit antibody was used to recognize 40 KDa C-gamma and C-alpha in vitro translation products. The specific aims of this proposal are designed to express C-gamma, C-beta, and C-alpha in E. Coli and mammalian cells and to characterize and compare them in vitro and in intact cells. The full-length human (h) C-gamma cDNA, as well as C-beta and C-alpha, will be expressed in E. Coli using the pT7-7 vector and the T7 promotor. The cDNAs will also be expressed in PKA deficient (Kin8) Y1 adrenal cells using the vectors pCMV-neo, which contains the cytomegalovirus promotor, and pZEM3-neo, which contains the metallothionine promotor. C-gamma will be analyzed for binding to the regulatory subunit, activation of the holoenzyme and phosphotransferase activity. The C-subunit isozymes will be compared regarding their peptide substrate inhibitor peptide specificity. In addition, C-gamma will be tested for the regulation of intact cell responses by reversion of a steroid synthesis deficiency in a PKA-deficient (Kin8) Y1 adrenal cell line. To test the role of C-gamma in regulating transcription, expression vectors containing the C-gamma coding sequence will be co-transfected with a fusion gene consisting of the enkephalin promotor coupled to a chloramphenicol acetyltransferase reporter gene. These studies should help elucidate potential differences among the C-subunit isozymes and determine if they have specialized roles in regulating specific cell functions.